This laboratory is involved in a complex effort to isolate heavy and light chain immunoglobulin messenger RNA from cells producing relatively small amounts of such proteins, and to characterize the relationship between coding sequences (exons) and functional units in the native immunoglobulin. The major objective of the research grant is related to preparation of the peptide fragments corresponding to coding sequences and to determination of how single amino acid changes in the hypervariable regions alter specificity. In addition, it has been a major objective to understand what controls proper assembly. During the past year, we systematically explored the effect of low levels of copper on the kinetics of chain assembly, because we had always suspected that kinetic variability in assembly was due to uncontrolled copper levels in the distilled water. We determined that we could produce elevated levels of HL half-molecules by careful control of copper levels. This turns out to be quite important because it will enable us to investigate the formation of hinge disulfides uncomplicated by formation of HL half-molecules. As previously reported, ds cDNA from the ARH cell line was inserted into pBR322 using a G-C tailing procedure. Two probes were used to screen for potential IgG mRNA sequences. For heavy chain sequences, a mouse gamma2b cDNA was used. A light chain probe was obtained from an immunoglobulin gene bank in Charon 28 (a generous gift from Dr. P. Leder); a 2.5Kb segment, containing the constant region for human k chain, was subcloned into pBR322 and M13, the latter being used to screen the ARH cDNA pBR322 clones. None of the cDNA clones contained inserts which hybridized to the gamma2b probe while two cDNA clones contained sequences complementary to the k chain probe. These contained inserts of less than 50bp and of approximately 300bp. The latter was subcloned into M13 and sequencing studies are in progress. In the coming year, we will attempt to isolate HL half molecules on molecular sieve columns and study the kinetics of their oxidation free of the complications of HL formation of unwanted immunoglobulins, such as IgE, which has been an objective from the start of this work. (AB)